Figure 4: Nicotinamide adenine dinucleotide increased intracellular Ca2+ levels and inhibition of Erk1/2 activity in cultured neurons. (a) Real-time trajectory of fluorescence intensity (n = 18). (b) Detection Ca2+ levels by adding MK-801 (40 μM) 200s before nicotinamide adenine dinucleotide treatment (n = 32). (c and d) nicotinamide adenine dinucleotide up-regulated the expression of p-Erk1/2 (n = 3). (e-i) The mRNA and protein expression levels of immediate-early response genes under different treatments, respectively. Inhibition of Erk1/2 activity blocked the nicotinamide adenine dinucleotide's effects on the expression of immediate-early response genes. Data in d were obtained by unpaired t-test. Data in panels (e and f, h and i) were obtained by one-way ANOVA combined with bonferroni post-test. *P < 0.05, **P < 0.01, ***P < 0.001 (n = 3).